Electrophoresis is an electrochemical process in which molecules with a net charge migrate in a solution under the influence of an electric current. It is a powerful technique for the separation and analysis of charged substances. A very important factor in the widespread use of electrophoresis is the utilization of stabilizing media such as polymer gels. This stabilizing media stabilizes the separation media (i.e., the liquid media through which the charged molecules migrate e.g., a buffer) against convection and flow which would otherwise disrupt separations. The microporous nature of stabilizers permits the electromigration of molecules through the stabilizers. However, because of the presence of particulates of various size and channels, stabilizers can cause zone broadening because of eddy migration and extensive adsorption between solutes and the stabilizer, for example. There is a desire to eliminate the use of such gels and adapt electrophoresis to on-line sample analysis, detection, quantification, and automated operations.
One automated version of electrophoresis that has been developed uses an open capillary tube, i.e., a tube typically made of fused silica containing a buffer without a stabilizing medium. In such a system, electrophoresis can take place with minimal interference and zone broadening can be minimized. The capillary acts like the microporous gel to counteract convective flow. Thus, the stabilizing effect of the capillary increases as the capillary diameter is decreased. This decrease in diameter of the capillary increases the surface to volume ratio and thus enhances heat dissipation.
In capillary electrophoresis, a buffer filled capillary is suspended between two reservoirs filled with buffer. Typical capillary diameters are less than 80 microns and typical lengths are more than one meter. An electric field is applied across the two ends of the capillary. Samples are introduced at the high potential end, which, under the influence of the electrical field, migrate toward the low potential end. When the samples leave the capillary zones after migrating through the capillary, they are detected by a detector.
In capillary zone electrophoresis (CZE), a sample zone is eluted by a carrier electrolyte. The carrier constituents have the same charge as the sample constituents to be separated. Separations are based upon differences in the electrophoretic mobilities of the constituents. Thus, significantly high resolution separations can be obtained and manipulated by changing the electrophoretic medium (e.g., pH and buffer composition).
Capillary zone electrophoresis provides a system for exploring the use of nonaqueous separation media. Furthermore, the elimination of a user gel or particulate system in the capillary enables the separation of large biomolecules and particulate-containing samples, such as viruses, cells, and organelles. CZE is limited, however, because of the limited availability of on-line detection methods. For further development and application of capillary zone electrophoresis, on-line electronic detection systems permitting good quantification are needed. Furthermore, there is a need for extremely sensitive detectors and particularly detectors of higher sensitivity than can be used on samples containing particulates.
Typically, ultraviolet-visible (UV-vis), fluorescent, or conductivity detection methods are used for detection of the species eluted in CZE. These detection methods have severe volume and sample size limitations, however, which present a major drawback in the use of CZE for the separation of complex mixtures. The ideal detectors for CZE should provide universal detection, selectivity, and sensitivity without degrading separation efficiency. Thus, mass spectrometry seems to be highly compatible with CZE.
In the on-line combination of CZE and mass spectrometry, the interface is typically accomplished by the application of electrospray ionization techniques. See, for example, J. A. Olivares et al., Anal. Chem., 59, 1230-1232 (1987). A strong electroosmotic flow in CZE (e.g., generally about 1 .mu.l/min), which results from a large potential applied across the capillary, is highly compatible with conventional mass spectrometers. The potential drop across the CZE column is typically .+-.30 kilovolts per meter. This potential drop is sufficiently large to result in the elution of ions, and to provide an electrospray. E. D. Lee et al., J. Chromatography, 458, 313 (1988) and Biomed. Environ. Mass. Spectrum., 18, 253 (1989) also disclose the successful coupling of CZE with mass spectrometry utilizing atmospheric pressure ion-spray interface, which is similar to electrospray ionization.
Although mass spectrometry is well-suited by analyzing eluates of capillary zone electrophoresis (CZE), a key element of the CZE-MS interface is the provision for electrical contact of the buffer at the capillary exit. Typically, a thick silver layer is deposited at the end of the capillary. Furthermore, in the application of CZE, the high voltage end is immersed in the sample and the opposite end is grounded as a part of the detection system. With a mass spectrometer, however, the detector end has to be inside a high vacuum chamber, and therefore cannot be easily grounded. Thus, there exists a need for a detection system in which good grounding is provided to the exit end of the capillary in a vacuum condition when CZE is interfaced with a mass spectrometer.